For dual-luciferase assay, the pKrox24(2xD-E_inD)Luc firefly luciferase reporter (28) was transfected together with the pTK-RL vector (Promega) in 3:1 (microgram of DNA) ratio. Both plasmids were transfected using FuGENE6 reagent, 0.75 μg of plasmid DNA per well of a 24-well plate containing 2 × 105 RCS cells, in 1:3.2 ratio of DNA/FuGENE6 (micrograms per microliter). The cells were treated with FGF2 for 24 hours, and the luciferase signals were determined using dual-luciferase assay (Promega). Briefly, cells were lysed in passive lysis buffer (100 μl per well) for 15 min, and 10 μl of lysate was used for determination of the dual-luciferase signal using the manufacturer’s protocol and TriStar Multimode Luminometer (Berthold Technologies); 40 μl of each luciferase substrate was used per sample, and the measurement times were 5 to 10 s for firefly and 5 s for Renilla luciferase.

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